Journal of the Taiwan Institute of Chemical Engineers 40 (2009) 1320

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Journal of the Taiwan Institute of Chemical Engineers

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Study of increasing lipid production from fresh water microalgae Chlorella vulgaris
Arief Widjaja a,*, Chao-Chang Chien b, Yi-Hsu Ju b
a
b

Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember, Kampus ITS Keputih Sukolilo, Surabaya, East Java 60111, Indonesia
Department of Chemical Engineering, National Taiwan University of Science and Technology, 43 Keelung Road, Section 4, Taipei 10607, Taiwan

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 6 March 2008
Received in revised form 30 June 2008
Accepted 1 July 2008

Study of increasing lipid production from fresh water microalgae Chlorella vulgaris was conducted by
investigating several important factors such as the effect of CO2 concentration, nitrogen depletion and
harvesting time as well as the method of extraction. The drying temperature during lipid extraction from
algal biomass was found to affect not only the lipid composition but also lipid content. Drying at very low
temperature under vacuum gave the best result but drying at 60 8C still retained the composition of lipid
while total lipid content decreased only slightly. Drying at higher temperature decreased the content of
triacylglyceride (TG). As long as enough pulverization was applied to dried algal sample, ultrasonication
gave no effect whether on lipid content or on extraction time. In addition to the increase of total lipid
content in microalgal cells as a result of cultivating in nitrogen depletion media, it was found that
changing from normal nutrient to nitrogen depletion media will gradually change the lipid composition
from free fatty acid-rich lipid to lipid mostly contained TG. Since higher lipid content was obtained when
the growth was very slow due to nitrogen starvation, compromising between lipid content and
harvesting time should be taken in order to obtain higher values of both the lipid content and lipid
productivity. As the growth was much enhanced by increasing CO2 concentration, CO2 concentration
played an important role in the increase of lipid productivity. At low until moderate CO2 concentration,
the highest lipid productivity could be obtained during N depletion which could surpassed the
productivity during normal nutrition. At high-CO2 concentration, harvesting at the end of linear phase
during normal nutrition gave the highest lipid productivity. However, by reducing the incubation time of
N depletion, higher lipid content as well as higher lipid productivity may still be achieved under this
condition.
2008 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords:
Chlorella vulgaris
Lipid
Nitrogen depletion
Harvesting time
Lipid productivity

1. Introduction
CO2 is recognized as the most important of the atmospheric
pollutants that contribute to the greenhouse effect. Reducing the
build-up of atmospheric CO2 can be accomplished by utilizing
photosynthetic organism which has ability to use CO2 for the
growing. Higher plants, e.g. trees, are capable to do this process.
However, these will not be enough to stabilize atmospheric CO2
levels sufciently to avoid a future greenhouse world (Benemann,
1997).
Microalgae is a photosynthetic microorganism that is able to
use the solar energy to combine water with carbon dioxide to
create biomass. Because the cells grow in aqueous suspension, they
have more efcient access to water, CO2, and other nutrients.

* Corresponding author. Tel.: +62 31 5946240; fax: +62 31 5999282.

E-mail address: arief_w@chem-eng.its.ac.id (A. Widjaja).

Microalgae, growing in water, have fewer and more predictable

process variables (sunlight, temperature) than higher plant
systems, allowing easier extrapolation from one site, even climatic
condition, to others. Thus, fewer site-specic studies are required
for microalgae than, for example, tree farming. Also, microalgae
grow much faster than higher plants and require much less land
areas. However, the utilization of microalgae to overcome global
warming is not enough without utilizing an algal biomass before
degradation.
Fatty acid methyl esters originating from vegetable oils and
animal fats are known as biodiesel. Biodiesel fuel has received
considerable attention in recent years, as it is a biodegradable,
renewable and non-toxic fuel. It contributes no net carbon dioxide
or sulfur to the atmosphere and emits less gaseous pollutants than
normal diesel (Antolin et al., 2002; Lang et al., 2001; Vicente et al.,
2004). High dependence on foreign oil, especially transportation
sector, gives rise to the importance of producing biodiesel for the
sake of national energy security.

1876-1070/$ see front matter 2008 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jtice.2008.07.007

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A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) 1320

There are four primary ways to make biodiesel, direct use and
blending, microemulsions, thermal cracking (pyrolysis) and
transesterication (Ma and Hanna, 1999). The most common
way is transesterication as the biodiesel from transesterication
can be used directly or as blends with diesel fuel in diesel engine
(Peterson et al., 1991; Zhang et al., 2003). Biodiesel, primarily
rapeseed methyl ester, has been in commercial use as an
alternative fuel since 1988 in many European countries (Lang
et al., 2001). However, in spite of the favourable impact that its
commercialization could provide, the economic aspect of biodiesel
production prevents its development and large-scale use, mainly
due to the high-feed cost of vegetable oil (Antolin et al., 2002; Lang
et al., 2001). Biodiesel usually costs over US $0.5/L, compared to US
$0.35/L for normal diesel (Zhang et al., 2003). Exploring ways to
reduce the high cost of biodiesel is of much interest in recent
biodiesel research, especially for those methods concentrating on
minimizing the raw material cost.
Microalgae have been suggested as very good candidates for
fuel production because of their advantages of higher photosynthetic efciency, higher biomass production and faster growth
compared to other energy crops (Dote et al., 1994; Ginzburg, 1993;
Miao and Wu, 2006; Milne et al., 1990; Minowa et al., 1995).
Microalgae systems also use far less water than traditional oilseed
crops. For these reasons, microalgae are capable of producing more
oil per unit area of land, compared to terrestrial oilseed crops.
Microalgae are very efcient biomass capable of taking a waste
(zero energy) form of carbon (CO2) and converting it into a highdensity liquid form of energy (natural oil).
Hundreds of microalgal strains capable of producing high
content of lipid have been screened and their lipid production
metabolism have been characterized and reported (Sheehan et al.,
1998). Most of them are marine microalgae. Recently, Miao and
Wu (Miao and Wu, 2004, 2006; Miao et al., 2004; Wu et al., 1992,
1994) reported a heterotrophic growth of Chlorella protothecoides
capable of yielding as high as 55% lipid content and converting the
lipid to biodiesel. Besides the need of expensive nutrient of
thiamine hydrochloride, the need of glucose instead of CO2 for
heterotrophic growth gain less interest in the view of global
warming issue. Using CO2 as carbon source, the strain yielded only
about 15% of lipid. Allard and Templier (2000) extracted lipid from
a variety of freshwater and marine microalgae and reported that
lipid content varied from 1 to 26%. A great deal of attention has
been focused on the autotrophic green microalga, Botryococcus
braunii, due to its high-hydrocarbon production level (Casadevall
et al., 1985; Metzger and Pouet, 1995; Wake and Hillen, 1980).
Sawayama et al. (1995) utilized this algae for continuously
operated CO2 xation and oil production. Despite the high-lipid
content of 64%, the growth rate of this strain was reported to be
very low (Sheehan et al., 1998).
Utilizing marine microalgal strains will give benet if large pond
is used for the system. As these strains can grow in brackish water,
that is, water that contains high levels of salt, they will not compete
for the land already being used by other biomass-based fuel
technologies. Freshwater microalgae, however, still can compete
with marine microalgae if, instead of using large pond, closed
photobioreactors which require less land area are used. Lower land
requirements were also assumed to be possible with the optical ber
devices (Sheehan et al., 1998). These kind of reactors are also able to
provide better dark and light photoperiod in the system as evidenced
from several experimental works using air lift bioreactor (Novakovic
et al., 2005), stirred tank photobioreactor (Huang and Rorrer, 2003)
and other types of closed photobioreactor (Rorrer et al., 1996; Rorrer
and Mullikin, 1999; Usui and Ikenouchi, 1997; Vernerey et al., 2001).
As productivity is measured in terms of biomass produced per day
per unit of available surface area, closed photobioreactors will give

much higher productivities. Although many reports are available

concerning the production of lipid from microalgae, as far as the
authors know, there is no report available for a thorough discussion
concerning the effect of growth conditions and extraction method
on the lipid productivity as well as on its content and composition.
These parameters are of vital importance for the application of the
system in larger scale.
Zhu et al. (2002) extracted fungal lipid of Mortierella alpina and
made comparison between wet biomass and biomass dried at
80 8C. Wet biomass was extracted according to the procedure of
Bligh and Dyer (1959) and dried biomass was extracted using
chloroform:methanol (2:1, v/v ratio). They found that dry
extraction was more effective than wet extraction. Solvent
extraction was still the main extraction method used by
researchers due to its simplicity and relatively inexpensive
requiring almost no investment for equipment (Letellier and
Budzinski, 1999). Inspite of many extraction methods developed
recently including supercritical or subcritical uid extraction, as
well as microwave and ultrasound assisted extraction, the yield of
the extraction of lipid from biomass using organic solvents,
including chloroform/methanol mixture, was still found superior
in comparison to supercritical uid extraction (Mendes et al.,
2006). Comparison between different methods to extract compounds with pharmaceutical importance from microalgae including Chlorella vulgaris was reported (Mendes et al., 2003) but no
comparison was made against chloroform/methanol solvent
extraction.
Much research was focused on the lipid trigger which refers to
the observation that under environmental stress, many microalgae
produce more lipid. It is generally accepted that the depletion of
the nitrogen from the medium induces lipid accumulation (Evans
and Ratledge, 1984b; Yoon and Rhee, 1983a). Botham and Ratledge
(1979) argued that the glucose conversion into lipids was
triggered, when nitrogen was exhausted, due to the high-energy
charge (ratio of ATP:AMP) present. The nitrogen source also was
reported to alter the amount of lipids (Evans and Ratledge,
1984a,b; Yoon and Rhee, 1983b). Turcotte and Kosaric (1988)
studied the biosynthesis of lipids on Rhodosporidium toruloides
ATCC 10788 under limiting amount of nitrogen required to trigger
lipid accumulation. The results showed that lipid accumulation
always started sometime after nitrogen reached a level of
3  10 5 M and the specic initial lipid productivity, expressed
as g/(L h) of storage lipid per g/L of lipid-free cellular material, was
constant. Sheehan et al. (1998) reported that the reason for the
increase of lipid content was that under nutrient starvation, the
rate of production of all cell components is lower, but oil
production remains higher, leading to an accumulation of oil in
the cells. Environmental stresses like nitrogen depletion lead to
inhibition of cell division, without immediately slowing down oil
production. They also suggested by controlling the timing of
nutrient depletion and cell harvesting, a net increase in total oil
productivity might be obtained.
The research aimed to produce lipid contained in fresh water
microalgae C. vulgaris using a 5-L closed fermentor. The effect of CO2
concentration, nitrogen concentration, harvesting time, and lipid
extraction method on the lipid content, lipid composition and lipid
productivity were investigated to obtain best condition under which
high-lipid content and high-lipid productivity could be achieved.
2. Materials and methods
2.1. Materials
A microalgal strain of C. vulgaris was kindly provided by Prof.
Hong-Nong Chou of The Institute of Fisheries Science, National

A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) 1320

Taiwan University. In addition to its easier growth in a relatively

cheap media without a necessity of utilizing very specic compound,
this strain was utilized since it gave signicant lipid content with
good growing rate as evidenced from our preliminary experimental
results and in comparison with other freshwater and seawater
microalgal strains conducted by Prof. Chou (data not shown). All
solvents and reagents were either of HPLC grade or AR grade. All
other chemicals used were obtained from commercial sources.
2.2. Medium and cultivation condition
The normal nutrition medium for cultivation of C. vulgaris was a
modication of Fitzgerald medium (Hughes et al., 1959) made by
adding 1 mL of each of IBI (a), IBI (b), IBI (c), IBI (d), and IBI (e) to 1 L
distilled water. IBI (a) contained, per 200 mL: NaNO3, 85.0 g;
CaCl2
2H2O, 3.70 g. IBI (b) contained, per 200 mL: MgSO4
7H2O,
24.648 g. IBI (c) contained, per 200 mL: KH2PO4, 1.36 g; K2HPO4,
8.70 g. IBI (d) contained, per 200 mL: FeSO4
7H2O, 1.392 g; EDTA
triNa, 1.864 g. IBI (e) contained, per 200 mL: H3BO3, 0.620 g;
MnSO4
H2O, 0.340 g; ZnSO4
7H2O, 0.057 g; (NH4)6Mo7O24
4H2O,
0.018 g; CoCl2
6H2O, 0.027 g; KBr, 0.024 g; KI, 0.017 g; CdCl2
(5/2)
H2O, 0.023 g; Al2(SO4)3(NH4)2SO4
24H2O, 0.091 g; CuSO4
5H2O,
0.00004 g; 97% H2SO4, 0.56 mL. This normal nutrition medium
resulted in a nitrogen content of 70.02 mg/L medium. The nitrogen
depletion medium was provided by eliminating the addition of IBI (a)
to result in a medium with a nitrogen content of 0.02 mg/L medium.

15

aerated at an air ow rate of 6 L/min with or without the addition

of pure CO2 gas. The fermentor is agitated at 100 rpm. Four pieces
of 18 W cool-white uorescent lamps are arranged vertically, at a
20-cm distance from the surface of fermentor to provide a
continuous light to the system. This gave an average light intensity
of 30 mE/(m2 s). The optical density of cells was measured at
682 nm every 24 h using UV-530 JASCO Spectrophotometer, Japan.
Cells were harvested at the end of linear phase, i.e. at a cell
concentration of about 1.1  107 cells/mL. To investigate the effect
of nitrogen depletion, 1 L of culture from the end of linear phase
was diluted by adding 3 L nitrogen depletion medium and the
cultivation continued for 7 and 17 d at which time the cells were
harvested and the lipid content as well as lipid productivity were
measured. Other conditions of incubation such as light intensity,
pure CO2 gas ow rate and temperature were all the same as the
corresponding normal nutrition condition.
2.6. Gas chromatography analysis
Sample was dissolved in ethyl acetate and 0.5 mL of this was
injected into a Shimadzu GC-17A (Kyoto, Japan) equipped with
ame ionization detector using DB-5HT (5% phenyl)-methylpolysiloxane non-polar column (15 m  0.32 mm ID); Agilent Tech.,
Palo Alto, California). Injection and detector temperature both
were 370 8C. Initial column temperature was 240 8C, and the
temperature was increased to 300 8C at a temperature gradient of
15 8C/min.

2.3. Lipid extraction

3. Results and discussion
Dry extraction procedure according to Zhu et al. (2002) as a
modication of the wet extraction method by Bligh and Dyer
(1959) was used to extract the lipid in microalgal cells. Typically,
cells were harvested by centrifugation at 8500 rpm for 5 min and
washed once with distilled water. After drying the samples using
freeze drier, the samples were pulverized in a mortar and extracted
using mixture of chloroform:methanol (2:1, v/v). About 50 mL of
solvents were used for every gram of dried sample in each
extraction step. After stirring the sample using magnetic stirrer bar
for 5 h and ultrasonicated for 30 min, the samples were centrifuged
at 3000 rpm for 10 min. The solid phase was separated carefully
using lter paper (Advantec lter paper, no. 1, Japan) in which two
pieces of lter papers were applied twice to provide complete
separation. The solvent phase was evaporated in a rotary
evaporator under vacuum at 60 8C. The procedure was repeated
three times until the entire lipid was extracted. The effect of
solvents having different polarities for extracting the lipid, as well
as the effect of drying temperature and ultrasonication time were
investigated in this study.
2.4. Effect of CO2 concentration
The effect of CO2 concentration on lipid content, lipid
composition and productivity was investigated by varying the
CO2 concentration. At rst, the culture was aerated under air ow
rate of 6 L/min without additional CO2. By taking into account the
CO2 content in air of about 0.03%, this condition resulted in about
2 mL/min CO2 as carbon source. The next batch was conducted
under the same air ow rate with the addition of 20, 50, 100, and
200 mL/min pure CO2 gas, or about 0.33, 0.83, 1.67, and 3.33% CO2,
respectively.
2.5. Effect of nitrogen concentration
At rst, cells of C. vulgaris was cultivated in 4 L normal nutrition
medium and incubated batchwisely at 22 8C. The system was

3.1. Effect of extraction method on lipid content and composition

Dry extraction procedure according to Zhu et al. (2002) and
Miao et al. (2004) was the main method employed to be
investigated in this study. Several important factors during the
extraction such as effect of solvent, drying temperature and
ultrasonication was investigated to see which method give the best
result in view of total lipid obtained, lipid composition and lipid
productivity.
3.1.1. Effect of solvent on the extraction
Instead of chloroform/methanol mixture, Miao and Wu (2006)
used hexane to extract lipid from C. protothecoides. Our results
using hexane as the solvent in the extraction of lipids resulted in
poor yield (data not shown) and chloroform/methanol mixture
was employed in this study.
3.1.2. Effect of drying temperature
Fig. 1 shows the effect of drying temperature on the lipid
content. Heating at 60 8C resulted in a slight decrease of lipid
content but when heating was conducted at 80 8C or higher
temperature, the lipid content decreased signicantly. Table 1
shows that the content of TG tend to decrease when higher
temperature was applied for the drying of algal sample. Oxidation
of fatty acid upon exposing to high temperature has been reported
(Oehrl et al., 2001). They reported that unsaturated fatty acid,
especially polyunsaturated free fatty acid (PUFA), was more
susceptible to oxidation than saturated fatty acid. The reasonable
explanation of the degradation of TG in Table 1 was the oxidation
of TG at high temperature for 12 h. The TG in microalgal cells
obtained in the present works should consist of highly unsaturated
fatty acids. Bockisch (1998) and Choe and Min (2007) also reported
that the degradation of TG by oxidation also resulted in the
formation of hydroperoxide group (OOH) in the chain. The
hydroperoxides formed can react further to aldehydes, ketones,

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A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) 1320
Table 2
Comparison of lipid composition under different ultrasonication time

Fig. 1. The effect of drying temperature on lipid content. Total lipid content was
calculated as w/w ratio of chloroform/methanol soluble fraction to dried algal
sample. Drying at 0 8C was provided by freeze drier, while drying at other
temperatures was conducted using oven for 12 h until all the water was removed,
i.e. nal water content of ca. 0%. Data are expressed as mean values  deviation
(n = 3).

Major lipid
components

Composition (%)
0 min

15  3 min

30  3 min

60  3 min

C16 FFA
C18 FFA
DG
TG
Others

1.36  1.4
2.74  0.2
5.4  0.1
71.53
18.97  1.1

0.30  0.3
2.96  0.2
5.09  0.2
69.11  1.2
22.84  1.2

0.70  0.1
1.83  0.1
5.32  0.1
79.07  1.3
13.08  1.3

0.41  0.4
1.99  0.1
5.03  0.1
75.11  0.9
17.87  1.1

and it was found that ultrasonication reduce the extraction

time without any signicant different in yield (data not shown).
It can be concluded from this result that most all lipids were
accumulated in the extracellular part of the microalgal cells and
sufcient pulverization is enough to help all the lipid extracted
from the cells without a necessity of applying ultrasonication to
obtain higher yield of lipid.
3.2. Effect of CO2 concentration, nitrogen depletion and harvesting
time on microalgal growth, lipid content, lipid composition and
productivity

Table 1
Comparison of lipid composition under different drying temperature
Major lipid
components

Composition (%)
0 8C

60 8C

80 8C

100 8C

C16 FFA
C18 FFA
DG
TG
Others

0.85  0.1
1.64  0.2
3.61  0.1
83.73  2.3
10.16  1.2

0.51  0.07
0.89  0.1
5.23  1.1
85.56  3.4
7.79  2.5

0.72  0.15
1.27  0.3
3.53  1.9
58.23  2.4
34.44  2.5

0.57  0.1
1.37  0.6
5.32  1.2
44.26  1.8
47.46  3.1

and fatty acids. The results in Table 1 in which the content of other
compounds was increased seemed to conrm the previous
observation. The others in Table 1 should therefore contained
aldehydes and ketones. The resistance of DG after drying may
indicate that they consisted of saturated fatty acids which were not
easily broken down in comparison to the highly unsaturated fatty
acid that may construct the TG.

3.2.1. Effect of CO2 concentration on growth

Fig. 3 shows the growth of algae under different CO2
concentration. At 15 d incubation time, the gure shows that
increasing CO2 ow rate until 50 mL/min enhanced the growth
tremendously. However, increasing CO2 ow rate further to 100
and 150 mL/min gave no increase on the growth (data not shown).
As shown in the gure, increasing to 200 mL/min CO2 ow rate
seemed to give even worse results by which until 2 d of incubation
time the growth rate was lower than that using 50 mL/min CO2.
The data obtained in the experimental results agreed with the data
reported by Riebesell et al. (2000) in which they found that
increasing CO2 concentration of up to 1% of air will increase lipid
produced by algae.
Sorensen et al. (1996) explained the mechanism of converting
CO2 to biomass as follows:

3.1.3. Effect of ultrasonication time

The effect of ultrasonication during extraction was investigated
and the results on total lipid content and lipid composition were
shown in Fig. 2 and Table 2, respectively. It can be seen from both
Fig. 2 and Table 2 that ultrasonication gave neither effect to the
increase of extracted lipid nor to the lipid composition. Extraction
without enough pulverization of dried algal sample was conducted

Fig. 2. Comparison of total lipid content under different ultrasonication time.

Solvent extraction was conducted three times each of which used different
ultrasonication time of 0, 15, 30 and 60 min as indicated in the gure. Data are
expressed as mean values  deviation (n = 3).

Using higher concentration of CO2 may result in decreasing the

pH since unutilized CO2 will be converted to H2CO3. On the other
hand, if there is not enough CO2 gas supply, algae will utilize
carbonate to maintain its growth. Since algae use CO2(aq) from
bicarbonate as a compensation of lacking enough CO2 from gas
supply, this will result in increase of pH. Table 3 shows the pH
range under different CO2 concentration. Higher CO2 ow rate
decreased the pH but during nitrogen starvation, the pH was
practically stable at around 7. As can be seen from Fig. 3, at CO2
ow rate of 200 mL/min, the growth was once very slow with pH
dropped to about 5. But, after 2 d, the growth increased greatly
indicating that the algae recovered from low pH due to exposing at
very high-CO2 concentration. At this condition, the pH was
monitored to increase from about 5 to 6.4 and constant around
this value which was the same pH range as that using lower CO2
ow rate. As the growth recovered at the same time during the

A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) 1320

Fig. 3. Comparison of growth curve under normal nutrition at an air ow rate of

6 L/min with the addition of pure CO2 gas at a ow rate of (*) 0 mL/min, (&)
20 mL/min, (^) 50 mL/min and (~) 200 mL/min. Optical density (OD) was
absorbance measured at 682 nm.

Table 3
Range of pH measured under different CO2 concentration
[CO2] (mL/min)

0
20
50
200

pH
Normal nutrition

N depletion

6.868.33
6.747.15
6.167.01
5.446.44

7.498.30
6.887.00
6.406.90
6.316.70

gradual increase of pH, it was evidenced from this result that the
microalgae C. vulgaris could survive under low pH albeit the
growth was slow. Iwasaki et al. (1996) reported the similar
behavior of a green algae Chlorococcum littorale in which under
sudden increase of CO2, activity of algae decreased temporarily and
then recovered after several days. The fact that C. vulgaris can
survive at a wide range of pH from 5 to above 8 was benecial in
considering of applying the algae in any conditions such as very
low pH under direct ue gas from power plant or higher pH when
exposed to not enough CO2 source.
3.2.2. Effect of nitrogen depletion and harvesting time
Fig. 4 shows the lipid content obtained at the end of linear phase
during normal nutrition and the results were compared with lipid
content obtained during nitrogen starvation using 20 mL/min CO2
ow rate. Period of incubation during normal nutrition was also

Fig. 4. Comparison of total lipid content during normal nutrition and nitrogen
starvation at CO2 ow rate of 20 mL/min. Incubation time under normal nutrition
was conducted for (&) 15 d and (&) 20 d. After normal nutrition, the medium was
changed to nitrogen depletion and the growth continued for 7 and 17 d. Total lipid
content was calculated as w/w ratio of chloroform/methanol soluble fraction to
dried algal sample. Data are expressed as mean values  deviation (n = 3).

17

Fig. 5. Comparison of lipid productivity during normal nutrition and nitrogen

starvation at CO2 ow rate of 20 mL/min. Incubation time under normal nutrition
was conducted for (&) 15 d and (&) 20 d. After normal nutrition, the medium was
changed to nitrogen depletion and the growth continued for 7 and 17 d. Oil
productivity was calculated as total lipid (mg) obtained per volume (L) of culture
per total incubation time (d) since the beginning of normal nutrition culture until
harvesting time. Data are expressed as mean values  deviation (n = 3).
Table 4
Typical information required to calculate lipid productivity
Parameters

Incubation time

Cell concentration (10 cells/mL)

Biomass/mL culture (mg/mL)
Total lipid content (%)
Lipid productivity (mg/(L d))

15 d

20 d

1.1
0.55
26.71
9.75

1.3
0.86
29.53
12.77

varied to investigate the difference. Fig. 4 shows that lipid content

obtained after 20 d was higher than that obtained after 15 d. This
was due to longer incubation time which led to less nitrogen
concentration in the medium. Fig. 4 also shows that longer time of
nitrogen starvation obviously resulted in higher accumulation of
lipid inside the cells.
Fig. 5 shows the lipid productivity calculated from the data in
Fig. 4. Typical calculation of productivity was given in Table 4. As
shown in this table, cell concentration obtained after 20-d
incubation was signicantly higher than that obtained after 15 d
which led to higher amount of dried algal sample for lipid
extraction. Higher lipid content and higher biomass obtained
resulted in higher productivity after 20-d normal nutrition period.
However, Fig. 4 also shows that after exposing to nitrogen
starvation condition for 17 d, lipid content were almost the same
for both batches. As a consequence, Fig. 5 shows that lipid
productivity obtained after 17-d nitrogen depletion was higher
since total time required for incubation was shorter. This 15-d
period of normal nutrition was employed for further investigation.
The results in Figs. 4 and 5 clearly show the two phenomena
occurred in this experimental results. The rst one was that lipid
content was increased by exposing to 7-d nitrogen starvation
condition and increased further under longer N starvation of 17 d.
The second phenomena was that lipid productivity was once
decreased by changing from normal nutrition to 7 d of nitrogen
depletion and then increased back again at longer nitrogen
starvation time.
Figs. 4 and 5 also reveals that higher lipid productivity can be
obtained by varying not only the length of nutrient starvation but
also the length of normal nutrition.

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A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) 1320

Table 5
Composition of major lipid components under different nutrient condition
Major lipid
components

Composition (%)
Normal
nutrition

7-D nitrogen
depletion

17-D nitrogen
depletion

C16 FFA
C18 FFA
DG
TG
Others

20.24  1.4
46.37  2.5
7.24  1.4
5.7  0.2
20.45

6.77  0.2
11.44  0.4
2.75  0.5
53.0  1.5
26.04  2.0

6.44  0.3
8.65  1.2
1.56  0.4
74.24  2.5
9.11  1.2

Lipid composition during normal nutrition and nitrogen

starvation was analyzed using gas chromatography and the results
were shown in Table 5. It is clear from the table that exposing to
longer nitrogen starvation resulted not only in increasing total
lipid content but also in increasing the content of TG. The lipid
composition gradually changed from free fatty acid-rich lipid to
TG-rich microalgal lipid with far less amount of impurities. As far
as we know, this is the rst report concerning the gradual change of
the lipid composition in microalgal cells upon exposing to nitrogen
starvation. In the viewpoint of converting the lipid for biodiesel
production, this changing is more favorable. Component of
others in Table 5 may represent low concentration of other
fatty acids, hydrocarbon, and bioactive compound which are
usually not valuable for direct production of biodiesel (Allard and
Templier, 2000). Isolation of these components, especially
important bioactive compound, however, may have signicant
economical benets which can support the economical value of the
whole biodisel production process.
3.2.3. Effect of CO2 concentration on lipid content and productivity
The effect of CO2 on growth as given in Fig. 3 correlates directly
to the lipid productivity since growth was enhanced tremendously
by increasing the CO2 concentration. Effect of CO2 concentration on
lipid content and lipid productivity were given in Figs. 6 and 7,
respectively.
As shown in Fig. 6, under all CO2 concentrations, the lipid
content tend to increase when the algae was exposed to nitrogen
starvation condition. Fig. 7 shows that during normal nutrition,
lipid productivity increased as CO2 ow rate was increased. Similar
with the results obtained in Fig. 5, exposing at nitrogen starvation
condition once resulted in decreasing the lipid productivity
although Fig. 6 shows that total lipid content was higher than
lipid obtained during normal nutrition (result of 7-d nitrogen
depletion). This was caused by the slow growth of algae under
nitrogen depletion. However, for 0 and 20 mL/min CO2, exposing at
longer time of nitrogen depletion (17 d) resulted not only in higher
lipid content but also in increasing the lipid productivity at about
the same or even higher than lipid productivity at the end of
normal nutrient.
At CO2 ow rate of 50 mL/min, lipid content after 17-d nutrient
starvation reached more than 50% which was the highest value
compared to lipid content using lower CO2 ow rate. Although the
lipid productivity under this condition cannot reach the same
value as that obtained under normal nutrition, however, this
condition may still be better due to its TG-rich lipid composition as
mentioned in the previous section. Furthermore, higher productivity than normal nutrition can still be achieved by shortening the
incubation time of less than 17 during nitrogen starvation.
This study investigated several important parameters responsible for increasing lipid content and lipid productivity from fresh
water microalgae C. vulgaris. The biomass and lipid productivity
obtained in the present work are not necessarily superior to those
reported elsewhere using different strain of microalgae. However,

Fig. 6. Effect of CO2 concentration and nitrogen depletion on total lipid content.
After using normal nutrition, the medium was changed to nitrogen depletion and
continued the growth for 7 and 17 d. The CO2 ow rate was ( ) 0 mL/min, (&)
20 mL/min, and (&) 50 mL/min. Total lipid content was calculated in the same way
as Fig. 2. Data are expressed as mean values  deviation (n = 3).

Fig. 7. Effect of CO2 concentration and nitrogen depletion on lipid productivity.

After using normal nutrition, the medium was changed to nitrogen depletion and
continued the growth for 7 and 17 d. The CO2 ow rate was ( ) 0 mL/min, (&)
20 mL/min, and (&) 50 mL/min. Oil productivity was calculated in the same way as
Fig. 3. Data are expressed as mean values  deviation (n = 3).

the simple and inexpensive method conducted in this study which

resulted in a signicant increase of lipid content while maintaining
the productivity at a high value can easily be applied to other strain
of microalgae. Improving of nutrient conditions such as the use of
spesic but inexpensive compounds which can increase the
growth, genetic engineering of DNA responsible for capability of
producing high content of lipid, as well as design of photobioreactor giving better performance of the fermentation process are
several other parameters that can be optimized. The study in the
present report is therefore expected to give one part of signicant
contributions for the strategy of developing biodiesel production
technology.
3.3. Concluding remark
Fresh water microalgae C. vulgaris was chosen as the subject of
investigating the lipid productivity due to its easy growth and its
signicant lipid content. Factors responsible for the increase of
lipid productivity such as CO2 concentration, nitrogen depletion,
harvesting time and extraction method were investigated.

A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) 1320

The drying temperature during lipid extraction from algal

biomass was found to affect both the lipid composition and the
lipid content. Drying using freeze drier will retain the original
composition of microalgal lipid, while higher temperature drying
decreased the content of TG. Drying at 60 8C still retained the
composition of lipid and only decreases slightly the lipid content.
No signicant effect was found in the effect of ultrasonication on
extraction. Sufcient pulverization is enough to help the entire
lipid extracted from the cells.
Higher lipid content was obtained by exposing to 7-d nitrogen
starvation condition and increased further under 17-d N
starvation. Interestingly, the lipid productivity was once
decreased under 7-d N depletion condition and then increased
back again under 17-d N starvation. Furthermore, it was found
that cultivating in nitrogen depletion media will result not only
in the accumulation of lipid in microalgal cells but also in gradual
changing in the lipid composition from free fatty acid-rich lipid
to lipid mostly contained TG. This new nding will be very
important in the viewpoint of further application for biodiesel
production.
Since accumulation of lipid occurs at nitrogen depletion
condition under which the growth was much slower or even no
growth was encountered, compromising between increasing lipid
content and harvesting time was necessary to obtain higher values
of both the lipid content and lipid productivity. CO2 concentration
played an important role in the increase of lipid productivity since
the growth was much affected by CO2 concentration. At 0 until
20 mL/min CO2 ow rate, the lipid productivity during nitrogen
depletion could be higher than that obtained at the end of linear
phase during normal nutrition. At higher CO2 concentration,
growth under normal nutrition gave higher lipid productivity.
However, by reducing the incubation time of N depletion, higher
lipid content as well as higher lipid productivity may still be
achieved under this condition. Higher lipid productivity can be
obtained by varying not only the length of nutrient starvation but
also the length of normal nutrition.
Acknowledgement
All of the experimental works of this research was funded by the
National Science Council of Taiwan. The authors expressed sincere
thanks for its support.
References
Allard, B. and J. Templier, Comparison of Neutral Lipid Prole of Various Trilaminar
Outer Cell Wall (TLS)-Containing Microalgae with Emphasis on Algaenan
Occurrence, Phytochemistry, 54, 369 (2000).
Antolin, G., F. V. Tinaut, Y. Briceno, V. Castano, C. Perez, and A. I. Ramirez, Optimisation
of Biodiesel Production by Sunower Oil Transesterication, Bioresour. Technol.,
83, 111 (2002).
Benemann, J. R., CO2 Mitigation with Microalgae Systems, Energy Conver. Manage.,
38 (Suppl.), S475 (1997).
Bligh, E. G. and W. J. Dyer, A Rapid Method of Total Lipid Extraction and Purication,
Can. J. Biochem. Physiol., 37, 911 (1959).
Bockisch, M., Fats and Oils Handbook, p. 98, AOCS Press, Champaign, Illinois, U.S.A.
(1998).
Botham, P. A. and C. Ratledge, A Biochemical Explanation for Lipid Accumulation in
Candida 107 and Other Oleaginous Microorganisms, J. Gen. Microbiol., 114, 361
(1979).
Casadevall, E., D. Dif, C. Largeau, C. Gudin, D. Chaumont, and O. Desanti, Studies on
Batch and Continuous Cultures of Botryococcus braunii: Hydrocarbon Production in
Relation to Physiological State, Cell Ultrastructure, and Phosphate Nutrition,
Biotechnol. Bioeng., 27 (3), 286 (1985).
Choe, E. and D. B. Min, Chemistry of Deep-Fat Frying Oils, J. Food Sci., 72, 77 (2007).
Dote, Y., S. Sawayama, S. Inoue, T. Minowa, and S. Yokoyama, Recovery of Liquid Fuel
from Hydrocarbon-Rich Microalgae by Thermochemical Liquefaction, Fuel, 73,
1855 (1994).
Evans, C. T. and C. Ratledge, Effect of Nitrogen Source on Lipid Accumulation in
Oleaginous Yeasts, J. Gen. Microbiol., 130, 1693 (1984a).

19

Evans, C. T. and C. Ratledge, Inuence of Nitrogen Metabolism on Lipid Accumulation by Rhodosporidium toruloides CBS 14, J. Gen. Microbiol., 130, 1705
(1984b).
Ginzburg, B. Z., Liquid Fuel (Oil) from Halophilic Algae: A Renewable Source of NonPolluting Energy, Renew. Energy, 3, 249 (1993).
Huang, Y. M. and G. L. Rorrer, Cultivation of Microplantlets Derived from the Marine
Red Alga Agardhiella subulata in a Stirred Tank Photobioreactor, Biotechnol. Prog.,
19, 418 (2003).
Hughes, E. O., P. R. Gorham, and A. Zehnder, Toxicity of a Unialgal Culture of
Microcystis Aeruginosa, Can. J. Microbiol., 4, 225 (1959).
Iwasaki, I., N. Kurano, and S. Miyachi, Effects of High-CO2 Stress on Photosystem II in a
Green Alga, Chlorococcum littorale, Which Has a Tolerance to High CO2, J.
Photochem. Photobiol. B: Biol., 36, 327 (1996).
Lang, X., A. K. Dalai, N. N. Bakhshi, M. J. Reaney, and P. B. Hertz, Preparation and
Characterization of Bio-Diesels from Various Bio-Oils, Bioresour. Technol., 80, 53
(2001).
Letellier, M. and H. Budzinski,
Microwave Assisted Extraction of Organic
Compounds, Analusis, 27, 259 (1999).
Ma, F. and M. A. Hanna, Biodiesel Production: A Review, Bioresour. Technol., 70, 1
(1999).
Mendes, R. L., B. P. Nobre, M. T. Cardoso, A. P. Pereira, and A. F. Palavra, Supercritical
Carbon Dioxide Extraction of Compounds with Pharmaceutical Importance from
Microalgae, Inorg. Chim. Acta, 356, 328 (2003).
Mendes, R. L., A. D. Reis, and A. F. Palavra, Supercritical CO2 Extraction of g-Linolenic
Acid and Other Lipids from Arthrospira (Spirulina)maxima: Comparison with
Organic Solvent Extraction, Food Chem., 99, 57 (2006).
Metzger, P. and Y. Pouet, N-Alkenylpyrogallol Dimethyl Ethers, Aliphatic Diol
Monoesters and Some Minor Ether Lipids from Botryococcus braunii: A Race,
Phytochemistry, 40 (2), 543 (1995).
Miao, X. and Q. Y. Wu, High Yield Bio-Oil Production from Fast Pyrolysis by Metabolic
Controlling of Chlorella protothecoides, J. Biotechnol., 110, 85 (2004).
Miao, X., Q. Wu, and C. Yang, Fast Pyrolysis of Microalgae to Produce Renewable
Fuels, J. Anal. Appl. Pyrol., 71, 855 (2004).
Miao, X. and Q. Y. Wu, Biodiesel Production from Heterotrophic Microalgal Oil,
Bioresour. Technol., 97, 841 (2006).
Milne, T. A., R. J. Evans, and N. Nagle, Catalytic Conversion of Microalgae and
Vegetable Oils to Premium Gasoline, with Shapeselective Zeolites, Biomass, 21,
219 (1990).
Minowa, T., S. Y. Yokoyama, M. Kishimoto, and T. Okakurat, Oil Production from Algal
Cells of Dunaliella tertiolecta by Direct Thermochemical Liquefaction, Fuel, 74,
1735 (1995).
Novakovic, G. V., Y. Kim, X. Wu, I. Berzin, and J. C. Merchuk, Air-Lift Bioreactors for
Algal Growth on Flue Gas: Mathematical Modeling and Pilot-Plant Studies, Ind.
Eng. Chem. Res., 44, 6154 (2005).
Oehrl, L. L., A. P. Hansen, C. A. Rohrer, G. P. Fenner, and L. C. Boyd, Oxidation of
Phytosterols in a Test Food System, JAOCS, 78 (11), 1073 (2001).
Peterson, C. L., M. Feldman, R. Korus, and D. L. Auld, Batch Type Transesterication
Process for Winter Rape Oil, Appl. Eng. Agric., 7 (6), 711 (1991).
Riebesell, U., A. T. Revill, D. G. Holdsworth, and J. K. Volkman, The Effects of Varying
CO2 Concentration on Lipid Composition and Carbon Isotope Fractionation in
Emiliania huxleyi, Geochim. Cosmochim. Acta, 64 (24), 4179 (2000).
Rorrer, G. L., C. Zhi, and M. P. Fuller, Development and Bioreactor Cultivation of a
Novel Semidifferentiated Tissue Suspension Derived from the Marine Plant Acrosiphonia coalit, Biotechnol. Bioeng., 49, 559 (1996).
Rorrer, G. L. and R. K. Mullikin, Modeling and Simulation of a Tubular Recycle
Photobioreactor for Macroalgal Cell Suspension Cultures, Chem. Eng. Sci., 54,
3153 (1999).
Sawayama, S., S. Inoue, Y. Dote, and S. Yokoyama, CO2 Fixation and Oil Production
Through Microalga, Energy Conver. Manage., 36 (69), 729 (1995).
Sheehan, J., T. Dunahay, J. Benemann, and P. Roessler, A Look Back at the U.S.
Department of Energys Aquatic Species Program-Biodiesel from Algae, Prepared
for U.S. Department of Energys Ofce of Fuels Development, by National Renewable Energy Laboratory, July (1998).
Sorensen, B. H., N. Nyholm, and A. Baun, Algal Toxicity Tests with Volatile and
Hazardous Compounds in Air-Tight Test Flasks with CO2 Enriched Headspace,
Chemosphere, 32 (8), 1513 (1996).
Turcotte, G. and N. Kosaric, Biosynthesis of Lipids by Rhodosporidium toruloides ATCC
10788, J. Biotechnol., 8, 221 (1988).
Usui, N. and M. Ikenouchi, The Biological CO2 Fixation and Utilization Project by
RITE(1). Highly-Effective Photobioreactor System, Energy Conver. Manage., 38
(Suppl.), 87 (1997).
Vernerey, A., J. Albiol, C. Lasseur, and F. Godia, Scale-Up and Design of a Pilot-Plant
Photobioreactor for the Continuous Culture of Spirulina platensis, Biotechnol. Prog.,
17, 431 (2001).
Vicente, G., M. Martinez, and J. Aracil, Integrated Biodiesel Production: A Comparison of Different Homogeneous Catalysts Systems, Bioresour. Technol., 92, 297
(2004).
Wake, L. V. and L. W. Hillen, Study of a Bloom of the Oil-Rich Alga Botryococcus
braunii in the Darwin River Reservoir, Biotechnol. Bioeng., 22 (8), 1637 (1980).
Wu, Q. Y., S. Yin, G. Y. Sheng, and J. M. Fu, A Comparative Study of Gases Generated
from Simulant Thermal Degradation of Autotrophic and Heterotrophic Chlorella,
Prog. Nat. Sci., 3, 435 (in Chinese) (1992).
Wu, Q. Y., S. Yin, G. Y. Sheng, and J. M. Fu, New Discoveries in Study on Hydrocarbons
from Thermal Degradation of Heterotrophically Yellowing Algae, Sci. China (B),
37, 326 (1994).

20

A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) 1320

Yoon, S. H. and J. A. Rhee, Quantitative Physiology of Rhodotorula glutinis for Microbial

Lipid Production, Process Biochem. (October), 2 (1983a).
Yoon, S. H. and J. S. Rhee, Lipid from Yeast Fermentation: Effects of Cultural Conditions
on Lipid Production and its Characteristics of Rhodotorula glutinis, J. Am. Oil Chem.
Soc., 60, 1281 (1983b).

Zhang, Y., M. A. Dube, D. D. McLean, and M. Kates, Biodiesel Production from Waste
Cooking Oil. 1. Process Design and Technological Assessment, Bioresour. Technol.,
89, 1 (2003).
Zhu, M., P. P. Zhou, and L. J. Yu, Extraction of Lipids from Mortierella alpina and Enrichment
of Arachidonic Acid from the Fungal Lipids, Bioresour. Technol., 84, 93 (2002).